Chromatin immunoprecipitation (ChIP) is an important technique used in the study of DNA/protein interactions. An advantage of ChIP is that it can be used for analysing the association of specific proteins, or their modified isoforms, with defined genomic regions. A review of existing ChIP technology is provided in O'Neill et al. (2003) “Immunoprecipitation of native chromatin, NChIP”, Methods: A Companion to Methods in Enzymology 31:76-82. ChIP may be used to determine whether proteins such as transcription factors and modified histones bind to a particular region on the endogenous chromatin of living cells or tissues.
In a ChIP assay, fragments of the DNA-protein complex (i.e. the chromatin) are prepared in such a way so as to retain the specific DNA-protein interactions. These chromatin fragments can then be immunoprecipitated using an antibody against the protein present in the complex. The isolated chromatin fraction can then be treated to separate the DNA and protein components. The identity of DNA fragments isolated in connection with a particular protein (i.e. the protein against which the antibody used for immunoprecipitation was directed) can then be determined by Polymerase Chain Reaction (PCR), Real Time PCR, hybridization on microarrays, direct sequencing or other technologies used for identification of DNA fragments of defined sequence.
Hence, a chromatin immunoprecipitation assay typically involves the following five key steps: (i) preparation of chromatin to be analysed from cells; (ii) immunoprecipitation of chromatin using an antibody; (iii) isolation of the precipitated chromatin fragments; (iv) DNA recovery from the precipitated product; and (v) DNA analysis.
The ChIP technique has two major variants that differ primarily in how the starting (input) chromatin is prepared. The first variant (designated NChIP) uses native chromatin prepared by micrococcal nuclease digestion of cell nuclei by standard procedures. The second variant (designated XChIP) uses chromatin cross-linked by addition of formaldehyde to growing cells, prior to fragmentation of chromatin, usually by sonication. Some workers have used mild formaldehyde cross-linking followed by nuclease digestion, and UV irradiation has been successfully employed as an alternative cross-linking technique.
Typically the immunoprecipitation of chromatin fragments is performed using an antibody specific to the protein of interest which is bound to DNA. The antibody-bound chromatin fragments may be isolated from the sample using a solid phase. For instance, the antibody itself may be directly linked to a solid phase such as agarose or magnetic beads which is then contacted with the chromatin. Alternatively, an antibody free in solution may be applied to the chromatin-containing sample, and then antibody-bound chromatin fragments isolated using an agent such as protein A, protein G or an anti-immunoglobulin antibody conjugated to the solid phase.
The solid phase which is used in this step is typically either a gel-type structure (usually based on the carbohydrate agarose) or magnetic beads (usually based on polymethacrylate type polymers). In either case the solid phase is dispersed within the liquid sample, and must be separated from the sample by some means after binding of the chromatin to the solid phase. A gel (e.g. agarose) can be spun down in a centrifuge to form a pellet which can then be separated from the liquid sample by aspiration. Magnetic beads are typically separated by using a magnet to pull the beads to the side of the vessel while the liquid sample is aspirated from the vessel.
In the case of agarose gels, the pellet formation and aspiration steps need to be repeated several times to remove all traces of the sample, which is inconvenient and slow. Although the use of magnetic beads is typically faster and more convenient than using agarose, the separation step using a magnet and aspiration still requires considerable skill and can be time-consuming. Moreover, both methods involve loss of product at various stages which can accumulate through the whole process. The handling steps necessary with gels and beads make it difficult to obtain high DNA recovery with good purity, as well as good reproducibility between assays. Agarose gels and magnetic beads are prone to non-specific binding of DNA and proteins, and it is difficult to provide adequate washing steps to reduce the resulting background signal. Thus standard ChIP assays typically require a large amount of sample while providing insufficient specificity in terms of the isolated DNA product. Such methods are also difficult to automate and unsuited to high throughput screening applications.
Thus there is a need for improved chromatin immunoprecipitation assay methods which address one or more of the above problems. In particular, there is a need for methods and products for isolating chromatin from a sample which are sensitive, specific and convenient to use.